Automated microscope platform with improved imaging and accurate neuron reconstruction capabilities for high-throughput studies of neuroregeneration

项目摘要/摘要 哺乳动物的中枢神经系统在受伤后通常不能再生，导致无法治愈的疾病。 背负着巨大的医疗负担。一个例外是一种称为损伤条件反射的显著效应，在这种效应中，损伤 神经元的外周纤维激活细胞过程，极大地促进神经再生。利用这一点 治疗的“有条件的”再生形式需要清楚地了解其潜在的机制， 尽管对哺乳动物系统进行了密集的研究，但仍然缺乏这种能力。具体地说，存在着知识鸿沟 关于神经元类型、形态和连通性对再生的影响。一种活体实验方法 线虫可以揭示飞秒激光条件再生的细胞机制 单个神经元纤维的外科手术和高精度显微镜。在蠕虫中发现的三个基因也 调节哺乳动物的损伤条件作用，证明这种方法可以发现关键保守的 机械装置。尽管这种方法在检查单个基因或机制方面是有效的，但它的手册 执行排除了它定义多个神经元类型和手术位置的再生能力。 因此，迫切需要加速成像和激光手术，以全面研究再生。 拟议项目的总体目标是优化自动显微镜平台并对其进行验证 通过广泛测试线虫中许多神经元类型的条件再生。这个项目的基本原理是 自动化平台将允许大规模的再生研究，这些研究目前是不切实际的，但需要 来完整地绘制再生路径。目标将通过以下具体目标实现：1)改进 图像对比度，以实现轴突的计算机可视化。2)开发实时机器学习方法 用于自动神经元重建。3)评估C. 优雅女装。Work for Aim 1将控制样本照明，并将新颖的实时图像处理应用于 改善神经元与其背景之间的对比度。在目标2中，这些改进的图像将是可逆的 压缩，计算增强，重建成神经元模型，并为手术做注释。在AIM 3，该集成平台将用于进行手术和对C. 研究关键基因在再生中的作用。拟议项目的创新方面包括： 用于病变条件处理的无脊椎动物模型、提高成像对比度的新光学方法和新的 用于实时神经元重建的机器学习技术。拟议方案的预期结果 研究是对基本的遗传和细胞机制的深刻洞察，这些机制决定了 执行条件再生和自动显微镜平台的高通量验证 成像和外科手术。这些结果意义重大，因为它们将建立重要的再生驱动因素 包括目前可以有效治疗的潜在治疗靶点 神经系统的不可治愈的损伤和疾病。

Project Summary/Abstract The mammalian central nervous system typically fails to regenerate after injury, leading to incurable conditions with immense healthcare burdens. An exception is a remarkable effect called lesion conditioning, where injury to a neuron’s peripheral fiber activates cellular processes to greatly enhance neuroregeneration. Exploiting this “conditioned” form of regeneration for therapy requires a clear understanding of its underlying mechanisms, which is still lacking despite intense research in mammalian systems. Specifically, there is a knowledge gap regarding the impact of neuron type, morphology, and connectivity on regeneration. An in vivo approach in the worm C. elegans can reveal the cellular mechanisms underlying conditioned regeneration by femtosecond laser surgery and high-precision microscopy of single neuronal fibers. Three genes identified in the worm also modulate mammalian lesion conditioning, demonstrating that this approach can discover key conserved mechanisms. Even though this approach is effective at examining single genes or mechanisms, its manual execution precludes it from defining regenerative capacity across multiple neuron types and surgery locations. Thus, there is a critical need to accelerate imaging and laser surgery to comprehensively study regeneration. The overall objectives of the proposed project are to optimize an automated microscope platform and validate it by broadly testing many neuron types in C. elegans for conditioned regeneration. The rationale for this project is that an automated platform will permit large-scale regeneration studies that are currently impractical but required to fully map regenerative pathways. The objectives will be achieved by the following Specific Aims: 1) Improve image contrast to permit computer visualization of neurites. 2) Develop a real-time machine learning approach for automated neuron reconstruction. 3) Assess regenerative capacity in a broad range of neuron types in C. elegans. Work for Aim 1 will control the sample illumination and apply novel, real-time image processing to improve the contrast between neurons and their background. In Aim 2, these improved images will be reversibly compressed, computationally enhanced, reconstructed into a neuron model, and annotated for surgery. In Aim 3, the integrated platform will be used to perform surgery and reimage neurites in many neuron types in C. elegans to examine the role of key genes in regeneration. Innovative aspects of the proposed project include: an invertebrate model for lesion conditioning, new optical methods for improving imaging contrast, and novel machine learning techniques for real-time neuronal reconstruction. The expected outcomes of the proposed study are deep insights into the fundamental genetic and cellular mechanisms that determine the ability to execute conditioned regeneration and the validation of an automated microscope platform for high throughput imaging and surgery. These results are significant because they will establish important drivers of regeneration in the central nervous system, including potential therapeutic targets that could effectively treat currently incurable injuries and diseases of the nervous system.